Transforming potentials of epidermal growth factor and nerve growth factor receptors inversely correlate with their phospholipase C gamma affinity and signal activation.

The exchange of nerve growth factor receptor/Trk and epidermal growth factor receptor (EGFR) phospholipase C gamma (PLC gamma) binding sites resulted in the transfer of their distinct affinities for this Src homology 2 domain-containing protein. Relative to wild-type EGFR, the PLC gamma affinity increase of the EGFR switch mutant EGFR.X enhanced its inositol trisphosphate (IP3) and calcium signals and resulted in a more sustained mitogen-activated protein (MAP) kinase activation and accelerated receptor dephosphorylation. In parallel, EGFR.X exhibited a significantly decreased mitogenic and transforming potential in NIH 3T3 cells. Conversely, the transfer of the EGFR PLC gamma binding site into the Trk cytoplasmic domain context impaired the IP3/calcium signal and attenuated the MAP kinase activation and receptor dephosphorylation, but resulted in an enhancement of the ETR.X exchange mutant mitogenic and oncogenic capacity. Our findings establish the significance of PLC gamma affinity for signal definition, the role of this receptor tyrosine kinase substrate as a negative feedback regulator and the importance of this regulatory function for mitogenesis and its disturbance in oncogenic aberrations.     

Phosphorylation sites at the C-terminus of the platelet-derived growth factor receptor bind phospholipase C gamma 1.

We have identified two tyrosine phosphorylation sites, Tyr 1009 and Tyr 1021, in the C-terminal noncatalytic region of the human platelet-derived growth factor (PDGF) receptor beta subunit. Mutant receptors with phenylalanine substitutions at either or both of these tyrosines were expressed in dog epithelial cells. Mutation of Tyr 1021 markedly reduced the PDGF-stimulated binding of phospholipase C (PLC) gamma 1 but had no effect on binding of the GTPase activator protein of Ras or of phosphatidylinositol 3 kinase. Mutation of Tyr 1009 reduced binding of PLC gamma 1 less severely. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, also reduced the PDGF-dependent binding of a transiently expressed fusion protein containing the two Src-homology 2 domains from PLC gamma 1. Mutation of Tyr 1021, or both Tyr 1009 and Tyr 1021, greatly reduced PDGF-stimulated tyrosine phosphorylation of PLC gamma 1 but did not prevent the tyrosine phosphorylation of other cell proteins, including mitogen-activated protein kinase. We conclude that Tyr 1021, and possibly Tyr 1009, is a binding site for PLC gamma 1.     

Pathway of phospholipase C activation initiated with platelet-derived growth factor is different from that initiated with vasopressin and bombesin

The mode of phospholipase C activation initiated with platelet-derived growth factor (PDGF) has been studied in comparison with that initiated with vasopressin and bombesin in a rat fibroblast line, WFB. Stimulation of WFB cells by PDGF, vasopressin, and bombesin elicites rapid hydrolysis of polyphosphoinositides and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). On stimulation by PDGF, there was a lag period of about 10 s before an increase in [Ca2+]i. No measurable lag period was observed in the [Ca2+]i response induced by vasopressin or bombesin. Pretreatment of WFB cells with phorbol 12-myristate 13-acetate profoundly inhibited inositol phosphate formation evoked by vasopressin and bombesin, but enhanced to some extent inositol phosphate formation stimulated by PDGF. In membranes prepared from WFB cells, GTP markedly augmented inositol polyphosphate formation induced by vasopressin and bombesin. It was not successful in showing the PDGF-stimulated formation of inositol phosphates in the membrane preparation. The effects of GTP, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) on polyphosphoinositide hydrolysis stimulated by growth factors were studied in WFB cells made permeable to nucleotides by treatment with either saponin or Pseudomonas aeruginosa cytotoxin. PDGF, vasopressin, and bombesin elicited inositol phosphate production in the permeabilized WFB cells in the absence of added GTP. GDP beta S, a competitive inhibitor of GTP-binding proteins (G-proteins), markedly reduced the bombesin- and vasopressin-stimulated production of inositol phosphates. However, the PDGF-stimulated production of inositol phosphates was not affected by the addition of GDP beta S. GTP gamma S, an agonist of G-proteins, largely enhanced the vasopressin- and bombesin-stimulated hydrolysis of inositol lipids when added at 10-100 microM. In the presence of GTP gamma S, the PDGF-stimulated hydrolysis of inositol lipids was not enhanced, but was reduced: 100 microM GTP gamma S reduced the stimulated hydrolysis to about a half of the control level. Only GTP gamma S, and no other nucleoside triphosphates, was found to have these effects. Activation of G-proteins in WFB cells by fluoroaluminate resulted in the inhibition of inositol phosphate production elicited with not only PDGF, but also with vasopressin and bombesin. These results indicate that a G-protein couples vasopressin and bombesin receptors to the activation of phospholipase C. Moreover, these results suggest that coupling of the PDGF receptor to phospholipase C is not mediated through a G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phospholipase D mimics platelet-derived growth factor as a competence factor in vascular smooth muscle cells.

Recent studies have suggested the importance of phosphatidylcholine (PC) metabolism in growth factor-stimulated cells. In these cells, PC is hydrolyzed not only by PC-specific phospholipase C but also by phospholipase D (PLD). In the present investigation, we show that the simple addition of PC-hydrolyzing PLD from Streptomyces chromofuscus to the culture medium of vascular smooth muscle cells elicits choline release into the medium accompanied by the formation of phosphatidic acid. In the presence of ethanol, this treatment elicits a formation of phosphatidylethanol (PEt) at the expense of phosphatidic acid. Furthermore, we show here that exogenous addition of S. chromofuscus PLD induces a marked DNA synthesis in quiescent vascular smooth muscle cells. This DNA synthesis induced by S. chromofuscus PLD is, like platelet-derived growth factor (PDGF)-elicited DNA synthesis, largely dependent on the presence of insulin. In addition, S. chromofuscus PLD-induced PEt formation and DNA synthesis were not affected by protein kinase C down-regulation, whereas PDGF-induced PEt formation and DNA synthesis were significantly inhibited. These observations strongly suggest that protein kinase-dependent activation of PLD is involved in mitogenic signal in PDGF-stimulated cells and that exogenously added PLD acts as a competence factor in the same way as PDGF.     

Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors.

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.     

Protein kinase C-independent expression of stromelysin by platelet-derived growth factor, ras oncogene, and phosphatidylcholine-hydrolyzing phospholipase C.

Changes in the expression of several genes play critical roles in cell growth and tumor transformation. A number of proteases are increased in some tumors, and the level of these enzymes correlates with the metastatic potential of several cancer cell lines. Stromelysin, with the widest substrate specificity, can degrade the extracellular matrix conferring metastatic potential to tumor cells. The mechanisms whereby growth factors and oncogenes control the expression of stromelysin are beginning to be characterized. In the study shown here we also identify a region in the stromelysin promoter which is involved in the induction of stromelysin in response to platelet-derived growth factor, phosphatidylcholine-hydrolyzing phospholipase C, and ras oncogene. Our results are consistent with the notion that platelet-derived growth factor/phosphatidylcholine-hydrolyzing phospholipase C induces stromelysin gene expression through a phorbol myristate acetate/protein kinase C-independent mechanism by acting through elements in the stromelysin promoter distinct from the 12-O-tetradecanoylphorbol-13-acetate-responsive element.     

Phospholipase D activation by the mitogens platelet-derived growth factor and 12-O-tetradecanoylphorbol 13-acetate in NIH-3T3 cells

The effect of mitogens on phospholipase D activity was investigated in NIH-3T3 fibroblasts by measuring the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity when 1-propanol acts as the phosphatidyl group acceptor. Platelet-derived growth factor (PDGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated phosphatidylpropanol production by the cells. The dose-response relationships for activation of phospholipase D and stimulation of thymidine incorporation by PDGF and TPA were comparable. The possibility that activation of phospholipase D is utilized by mitogens as a trans-membrane pathway for signalling cell growth is discussed.     

Characterization of baculovirus-expressed human alpha and beta platelet-derived growth factor receptors.

In an effort to biochemically characterize PDGF receptors and their mechanism of activation, recombinant baculovirus vectors containing the cDNAs of the human alpha PDGF receptor or beta PDGF receptor were engineered. Characterization of recombinant PDGF receptor expression in infected Sf9 insect cells by immunoblot analysis with specific PDGF receptor peptide antisera revealed that the alpha and beta PDGF receptor gene products were translated as 160- and 165-kDa transmembrane proteins, respectively. Ligand binding analysis demonstrated saturable, high-affinity binding of either 125I-labeled PDGF AA or 125I-labeled PDGF BB to Sf9 cells expressing the recombinant alpha PDGF receptor. In contrast, recombinant beta PDGF receptor expressing Sf9 cells showed high-affinity binding only for PDGF BB. Analysis of the kinetics of PDGF receptor expression demonstrated that receptor number increased dramatically from 24- to 48-h postinfection. Early in infection, the PDGF receptors were present in low numbers, lacked tyrosine phosphorylation, and exhibited ligand-dependent tyrosine phosphorylation. However, with increasing time postinfection and increasing receptor number, the PDGF receptors became constitutively tyrosine-phosphorylated in serum-free culture medium. Cross-linking studies revealed that receptor activation involved ligand-independent receptor dimer formation at high receptor number. Thus, these results strongly suggest that PDGF stabilizes and increases the frequency of PDGF receptor interaction, which ultimately results in PDGF receptor activation and intracellular signaling.     

Phosphatidic acid modulates DNA synthesis, phospholipase C, and platelet-derived growth factor mRNAs in cultured mesangial cells. Role of protein kinase C

Increases in cell phosphatidic acid content occur in response to a wide variety of agonists, many of which have growth promoting properties. These changes have correlated with calcium flux, enzyme activation, gene induction, or cell proliferation. In the current studies we show that exogenous phosphatidic acid (PA) and phosphatidylserine stimulate phosphoinositide hydrolysis and DNA synthesis in cultured human renal mesangial cells. These phospholipids also induce mRNAs for platelet-derived growth factor (PDGF). The activation of phospholipase C by PA appears to be desensitized via protein kinase C as brief preincubation with phorbol ester abrogates the effect. PA-induced DNA synthesis is only partly mediated via protein kinase C as co-incubation with the inhibitor staurosporine blunts DNA synthesis by only one-third. In contrast, induction of PDGF A-chain mRNA is almost totally inhibited by staurosporine. We propose that changes in endogenous phospholipids such as PA or phosphatidylserine may serve as common signaling pathway for a variety of growth factors. Induction of PDGF proto-oncogenes via protein kinase C may represent one mechanism by which this cell activation occurs.     

Differential expression of platelet-derived growth factor receptors in human malignant glioma cell lines.

Glioma cells in culture express platelet-derived growth factor (PDGF) A- and B-chains and secrete PDGF-like activity that is mainly PDGF-AA. In this work, we show that the PDGF alpha- and beta-receptors are independently expressed in human malignant glioma cells. We also define three different receptor phenotypes that are related to the morphology of glioma cells: cells with only alpha-receptors, only beta-receptors, or with both types of receptors. By the help of Northern blot analyses, 125I-PDGF-binding experiments, and immunoprecipitations the receptors are shown to be structurally normal PDGF receptors, except for minor variations in size that probably are due to differences in glycosylation. PDGF-BB induces DNA synthesis in cells of all three receptor phenotypes, whereas PDGF-AA or PDGF-AB has this effect only on cells with alpha- or with alpha- and beta-receptors. 125I-PDGF-AB binds with high affinity and down-regulates beta-receptors only in cells where alpha-receptors are present in addition to beta-receptors. Thus, the different functional capacities of PDGF isoforms on glioma cells fit with their known receptor-binding specificities and are compatible with the hypothesis that the isoforms act by inducing dimeric receptor complexes. When data on PDGF A- and B-chains, as well as alpha- and beta-receptor expression are compiled and the pattern of receptor binding specificity is taken into account, the majority of glioma cell lines are found to have a phenotype that makes autocrine stimulation possible.     

Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation

Platelet-derived growth factor (PDGF) was found to induce dimerization of purified B-type PDGF receptors, as analyzed by sodium dodecyl sulfate gel electrophoresis after covalent cross-linking using disuccinimidyl suberate. PDGF-BB was 20-fold more effective than PDGF-AB; PDGF-AA was without effect. The dimerization was dose-dependent and was maximal at 0.5-2 micrograms/ml PDGF-BB; at higher concentrations dimerization was less abundant. This indicates that dimerization occurred when one PDGF-BB molecule bound two receptor molecules. The dimerization correlated to activation of the tyrosine kinase of the receptor, determined as autophosphorylation, but was not dependent on phosphorylation reactions because it occurred also in the absence of ATP. Furthermore, dimerization of the receptor correlated with the ability to phosphorylate phosphofructokinase, an exogenous substrate. The complex of ligand and receptor dimer was stable; it resisted electrophoresis under nondenaturing conditions, as well as gel chromatography. The present data indicate that intermolecular mechanisms are involved in signal transduction from the external ligand binding domain to the internal effector domains of the B-type PDGF receptor.     

A protein that is highly related to GTPase-activating protein-associated p62 complexes with phospholipase C gamma.

p62 is a highly tyrosyl phosphorylated protein that was first identified in immunoprecipitates of the GTPase-activating protein (GAP) of p21ras from cells transformed by oncogenic nonreceptor tyrosine kinases or stimulated through tyrosine kinase receptors (C. Ellis, M. Moran, F. McCormick, and T. Pawson, Nature 343:377-381, 1991). In this article we describe a highly related 62-kDa protein that becomes tyrosyl phosphorylated and associated with phospholipase C gamma (PLC gamma) in C3H10T1/2 cells stimulated with epidermal growth factor (EGF) or transformed by v-src. GAP-associated and PLC gamma-associated p62 comigrated in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited nearly identical phosphotryptic peptide patterns. That the association of p62 with PLC gamma was direct and not mediated through binding of GAP-p62 to PLC gamma or to the EGF receptor (and coprecipitation of the receptor with PLC gamma) was demonstrated by (i) the inability to detect GAP in PLC gamma immunocomplexes or PLC gamma in GAP immunocomplexes, (ii) the association of p62 with PLC gamma in v-src-transformed cells in the absence of EGF stimulation, and (iii) in vitro solution binding and direct blotting of p62 with a glutathione S-transferase fusion protein containing the Src homology 2 (SH2) domains of PLC gamma. Unlike GAP, whose N-terminal SH2 mediates the interaction between GAP and p62, PLC gamma was found to require both its N- and C-terminal SH2 regions for p62 binding. These studies demonstrate that a protein identical to or highly related to GAP-associated p62 binds PLC gamma and suggest a means by which "cross-talk" between PLC gamma- and GAP-mediated signalling may occur.     

Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C.

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.     

Tyrosine phosphorylation of Grb2-associated proteins correlates with phospholipase C gamma 1 activation in T cells.

Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.     

Phospholipase C-protein kinase C mediated phospholipase D activation pathway is involved in tamoxifen induced apoptosis

Tamoxifen (TAM) is the endocrine therapeutic agent the most widely used in the treatment of breast cancer, and it operates primarily through the induction of apoptosis. In this study, we attempted to elucidate the non-ER mediated mechanism behind TAM treatment, involving the phospholipase C-protein kinase C (PLC-PKC) mediated phospholipase D (PLD) activation pathway, using multimodality methods. In TAM treated MCF7 cells, the PLC and PLD protein and mRNA levels increased. Phosphatidylethanol (PEt) and diacylglycerol (DAG) generation also increased, showing increased activity of PLD and PLCgamma1. Translocation of PKCalpha, from cytosol to membrane, was observed in TAM treated cells. By showing that both PKC and PLC inhibitors could reduce the effects of TAM-induced PLD activation, we confirmed the role of PKC and PLC as upstream regulators of PLD. Finally, we demonstrated that TAM treatment reduced the viability of MCF7 cells and brought about rapid cell death. From these results, we confirmed the hypothesis that TAM induces apoptosis in breast cancer cells, and that the signal transduction pathway, involving PLD, PLC, and PKC, constitutes one of the possible mechanisms underlying the non-ER mediated effects associated with TAM.     

Dissociation of tyrosine phosphorylation and activation of phosphoinositide phospholipase C induced by the protein kinase C inhibitor Ro-31-8220 in Swiss 3T3 cells treated with platelet-derived growth factor

Platelet-derived growth factor (PDGF) stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (Ptd InsP₂) via phospholipase C-γ1 (PLC-γ1) in Swiss 3T3 cells. Treatment of cells with the protein kinase C (PKC) inhibitor Ro-31-8220 greatly decreased PDGF-induced tyrosine phosphorylation of PLC-γ1, but paradoxically enhanced the production of inositol phosphates (InsPs). The inhibitor also caused an increase of PDGF receptor tyrosine phosphorylation at later times. The changes in phosphorylation of the receptor were correlated with alterations in PLC-γ1 translocation to the particulate fraction. Thus, although activation of PLC-γ1 was associated with phosphorylation of the receptor and translocation of the enzyme to the particulate fraction, it was dissociated from its tyrosine phosphorylation. A non-receptor-associated, cytosolic tyrosine kinase also was found to phosphorylate PLC-γ1 in a PDGF-dependent manner, but was not inhibited by Ro-31-8220 in vitro. PKC depletion by phorbol ester treatment decreased the tyrosine phosphorylation of PLC-γ1 induced by PDGF and slowed the translocation of PLC-γ1, but Ro-31-8220 produced further effects. The effect of Ro-31-8220 to enhance the production of InsPs could not be attributed to inhibition of PKC since InsPs production with PDGF was decreased in PKC-depleted cells and a stimulatory effect of the inhibitor was still evident. Interestingly, Ro-31-8220 decreased the radioactivity in phosphatidylinositol and increased that in phosphatidylinositol 4-phosphate and PtdInsP₂ in cells labeled with myo[³H]inositol. The increased synthesis of PtdInsP₂ could contribute to the increased production of InsPs induced by Ro-31-8220. In summary, these results support the conclusion that the activation of PLC-γ1 in response to PDGF requires autophosphorylation of the receptor and membrane association of PLC-γ1, but not phosphorylation of the enzyme. Furthermore, the effects of Ro-31-8220 on the tyrosine phosphorylation and activity of PLC-γ1, and on PtdInsP₂ synthesis cannot be attributed to inhibition of PKC. © 1997 Elsevier Science B.V. All rights reserved.     

Ligand-induced interaction between alpha- and beta-type platelet-derived growth factor (PDGF) receptors: role of receptor heterodimers in kinase activation.

Two types of PDGF receptors have been cloned and sequenced. Both receptors are transmembrane glycoproteins with a ligand-stimulatable tyrosine kinase site. We have shown earlier that ligand-induced activation of the beta-type PDGF receptor is due to the conversion of the monomeric form of the receptor to the dimeric form [Bishayee et al. (1989) J. Biol. Chem. 264, 11699-11705]. In the present studies, we have established the ligand-binding specificity of two receptor types and extended it further to investigate the ligand-induced association state of the alpha-receptor and the role of alpha-receptor in the activation of beta-receptor. These studies were conducted with cells that express one or the other type of PDGF receptor as well as with cells that express both types of receptors. Moreover, ligand-binding characteristics of the receptor were confirmed by immunoprecipitation of the receptor-125I-PDGF covalent complex with type-specific anti-PDGF receptor antibodies. These studies revealed that all three isoforms of PDGF bind to alpha-receptor, and such binding leads to dimerization as well as activation of the receptor. In contrast, beta-receptor can be activated only by PDGF BB and not by PDGF AB or PDGF AA. However, by using antipeptide antibodies that are specific for alpha- or beta-type PDGF receptor, we demonstrated that in the presence of alpha-receptor, beta-receptor kinase can be activated by PDGF AB. We present here direct evidence that strongly suggests that such PDGF AB induced activation of beta-receptor is due to the formation of a noncovalently linked alpha-beta receptor heterodimer.     

Ligand activation causes a phosphorylation-dependent change in platelet-derived growth factor receptor conformation

The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.     

Partial purification and characterization of a growth factor present in goat's colostrum. Similarities with platelet-derived growth factor.

A factor in goat's colostrum which stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts has been purified approx. 350-fold by a sequence of acid precipitation, cation-exchange chromatography and gel filtration. The growth factor is a highly basic, heat stable (100 degrees C for 5 min) polypeptide with Mr approx. 35000. The polypeptide resists denaturation by guanidinium chloride or urea but is totally inactivated by treatment with reducing agents. The factor, which we have termed colostric basic growth factor ( CBGF ), inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 fibroblasts but does not inhibit 125I-EGF binding to epidermoid A431 cells. CBGF interacts synergistically with plasma in stimulating DNA synthesis in quiescent Swiss 3T3 cells. The chemical and biological properties of CBGF are thus very similar to the properties reported for the human platelet-derived growth factor. Although high concentrations of CBGF are present in the colostrum of goats, cows, and sheep, the milk of these species contains little or no factor. The origin and possible functions of CBGF are unknown.